Novel assay for detecting immune responses involving antigen specific cytokine and/or antigen specific cytokine secreting T-cells

ABSTRACT

Here, we describe a sensitive and specific assay and kit for the detection of chemokines having activity that is upregulated by Th−1 cytokines (such IFN-γ) and chemokines that upregulate the activity of Th−1 cytokines (such as IFN-γ). In a typical embodiment, detection of the chemokine monokine induced by gamma interferon (MIG) provides a measure of the biological effect of IFN-γ rather than direct quantitation of IFN-γ or IFN-γ secreting cells per se. Upregulation of MIG expression was observed following in vitro activation of PBMC with defined CD8 +  T cell epitopes derived from influenza virus, CMV, or EBV, and in all cases this was antigen-specific, genetically restricted and dependent on both CD8 +  T cells and IFN-γ. Responses as assessed by the MIG assay paralleled those detected by conventional IFN-γ ELISPOT, but the magnitude of response and sensitivity of the MIG assay were superior. Our data validate this novel method for the detection of high as well as low levels of antigen-specific and genetically restricted IFN-γ activity or MIG.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] Here, we describe a sensitive and specific assay and kit for thedetection of chemokines having activity that is upregulated by Th−1cytokines such as IFN-γ (interferon-γ) and chemokines that upregulatethe activity of Th−1 cytokines (such as IFN-γ). The invention alsorelates to a sensitive and novel immunoassay method and kit fordetecting MIG (Monokine Induced by interferon-γ) or other chemokineswhose production is upregulated by IFN-γ. Also, the invention relates toa method of assessing the effectiveness of a compound or system ininducing an immune response by detecting the induction of the expressionof such a chemokine. All references mentioned in this application areincorporated herein by reference, in their entirety.

[0003] 2. Description of the Background Art

[0004] The ability to assess specific immune responses is critical forunderstanding the immune mechanisms underlying disease, defining thetypes of responses to be induced by vaccination, and evaluating vaccineefficacy. Particular emphasis has been placed on the measurement of Tcell mediated responses and the identification of immune markers thoughtto correlate with protection. In this specification and the claims thatfollow, the term “immune response” includes responses that modify apre-existing immune response.

[0005] Cytokines are immune system proteins that are biological responsemodifiers. Monokines are chemokines secreted from monocytes. Chemokinesare cytokines that have chemoattractant properties. These biologicalproteins are discussed, for example, in the Illustrated Dictionary ofImmunology, ed. Cruse, J. M. and Lewis, R. E., CRC Press, N.Y., (1995).

[0006] In the present application and the claims that follow, animmunoreactive substance is defined as a substance to which antibodies,or immune cells, (such as T cells and NK cells) can bind, or whichstimulates the production of antibodies or activate or induces T cells.That is, an immunoreactive substance can be considered as a compoundcapable of inducing an immune response. Although immunoreactivesubstances are typically thought of as whole molecules or organisms, theterm “immunoreactive substance” may also be considered as that portionof a molecule or organism which elicits the immune response. For thepurposes of the present specification and the claims that follow, twoimmunoreactive (including antigenic) substances are considered to be thesame if they each share at least one common immunoreactive portion.

[0007] IFN-γ is a prototypic Th−1 cytokine produced by a variety ofcells including CD4⁺ T cells, CD8⁺ T cells and NK cells (1). Theimportance of this cytokine in mediating protection against a number ofpathogens, including parasites, bacteria, and viruses has been wellestablished (1). IFN-γ has been known to play a central role inorchestrating a range of immunological programs which are critical forimmune protection. These programs include induction of genes involved inantigen processing, upregulation of MHC Class I and Class II expression,induction of oxygen and nitrogen radicals, and stimulation of chemokineproduction in vitro and in vivo (1-5). Thus, in many systems, detectionof IFN-γ or IFN-γ secreting cells serves a marker for the biologicaleffects of IFN-γ activity. Accordingly, in many systems, detection ofIFN-γ or IFN-γ secreting cells following exposure to antigen isfrequently used to determine immunological responsiveness.

[0008] Through their ability to recruit distinct populations ofleukocytes, chemokines have the ability to enhance antigen-specificimmune responses. Since IFN-γ is known to regulate the production ofvarious chemokines (6), we sought to determine if one or more chemokinescould be used as a surrogate marker for antigen-specific IFN-γproduction. We hypothesized that evaluating the biological effects ofIFN-γ production rather than directly quantitating IFN-γ or IFN-γproducing cells per se may provide a more sensitive and reproduciblemeans of detecting antigen-specific IFN-γ activity. Accordingly, westudied a panel of chemokines implicated in IFN-γ mediated immuneresponses, including Monokine Induced by interferon-γ (MIG),Interferon-γ-inducible Protein-10 (IP-10), Monocyte ChemoattractantProtein-1 (MCP−1), Macrophage Inflammatory Protein-α (MIP-α), andRegulated Upon Activation, Normal T-cell Expressed and Secreted (RANTES)(7,8). This disclosure describes a novel assay for detectingantigen-specific MIG or antigen-specific IFN-γ or antigen-specific IFN-γproducing T-cells, based on flow cytometric quantitation of theantigen-specific, MHC-restricted, IFN-γ mediated induction of MIGexpression. Our studies establish that this is a specific and sensitiveassay for detecting high as well as low levels of antigen-specificIFN-γ- and/or antigen-specific IFN-γ secreting T-cells.

[0009] To date, the detection of low levels of antigen-specific cellularimmune responses has been problematic, particularly in human systems.Antigen-responsive CD4⁺ T cells responses are routinely detected byassessment of lymphoproliferative potential or capacity to produceantigen-specific cytokines via ELISA. Antigen-specific CD8⁺ T cellresponses are generally still evaluated by cytotoxic lysis of targetcells or limiting dilution analysis (LDA), as they have been for over 15years. However, these conventional assays are cumbersome and laborious,require extensive tissue culture and are not sensitive enough to detectlow frequencies of antigen-specific cells. Furthermore, they can not beused to evaluate responses associated with cells which may respond tospecific antigen by cytokine production, for example, but may notproliferate.

[0010] More recently, alternative methods have been developed to detectantigen-specific CD4⁺ and CD8⁺ T cell mediated immune responses. Theseassays include enumeration of cytokine producing cells at the singlecell level by ELISPOT or by flow cytometry using intracellular cytokinestaining techniques, or by directly quantitating peptide-specificclonotypes using tetramer technology.

[0011] In certain applications, it is desirable to evaluate theproduction of specific cytokines produced by antigen-specific cells.Currently, detection of antigen-specific cytokine-producing cells bycytokine-specific ELISPOT assay is gaining widespread acceptance as themost appropriate method available for detecting antigen-specificcellular immune responses. This method determines the number of cellsproducing a specific cytokine after in vitro culture in the presence ofa specific peptide/immunogen and can be used to reproducibly detect lownumbers of cytokine producing cells. The sensitivity for detecting lowfrequencies of responsive cells can be increased with prolonged cultureand/or restimulation. However, prolonged in vitro cultivation andvariations of culture conditions may not accurately reflect in vivoimmunologically relevant events. Additionally, the laboratory procedurefor completion of the ELISPOT assay is time consuming. Furthermore,quantitation of ELISPOT is achieved by subjective manual counting or bycomputerized counting microscopes which provide an objective andreproducible means of enumerating ELISPOTs, however, the cost of thesemachines do not make them readily available to most laboratories.Moreover, ELISPOT assays require additional time for coating plates andtypically longer culture times and an additional day for development forthe assay. Accordingly, there is a need in the art for a sensitive,specific, and relatively faster assay that requires short culture timeprior to analysis.

[0012] Tetramer staining is more sensitive than traditional methods fordetecting antigen-specific cells, but the use of this technique isrestricted to well characterized epitopes in association with definedMHC alleles, and requires subsequent culture for functionalcharacterization of tetramer-positive cells.

[0013] Flow-based intracellular staining methods provide a technicallysimple and relatively fast method for identifying antigen-responsivecells; however, it is difficult to detect low numbers ofantigen-specific cytokine-producing cells at levels significantly abovebackground. The limitation of the assay lies in the fact that lowfrequency of cytokine producing cells may be indistinguishable abovebackground levels. Addition of immune enhancer reagents, such asantibodies to CD28, are frequently used to augment costimulation inculture conditions but these reagents may bias the results. Success withintracellular staining assays have been most frequently reported instudies evaluating CD4⁺ T cell responses to viral antigens using PBMCfrom chronically infected individuals (for example, CMV or HIV). Verylimited success has been reported for the detection of ex vivoantigen-specific cells in CD4⁺ or CD8⁺ T cells from immunizedindividuals where the number of circulating antigen-specific cells maybe considerably lower than that found in individuals exposed to theinfectious agent. Indeed, without using tetramers to select asubpopulation of specific cells for evaluation, it has not been possibleto detect low frequencies of antigen-specific cells using flow cytometryfor intracellular cytokines. Thus, there is a need in the art for a flowcytometric method of detection low levels of antigen-specific cells withreadily available reagents.

[0014] In the assays described above, antigen-specific IFN-γ-immuneresponsiveness is evaluated directly by detecting IFN-γ or IFN-γproducing cells; these assays do not measure the biological response ofIFN-γ production from antigen-specific cells as is possible through theamplification effect of MIG. This results in an inability of IFN-γELISPOT assays and other detection methods to detect antigen-specificIFN-γ responses at low levels. Accordingly, there is a need in the artfor a very sensitive assay for quantitative measure of IFN-γ activity orfor detecting antigen-specific IFN-γ-producing cells.

[0015] Farber et al., WO 92/10582, published Jun. 25, 1992, teachmethods for producing MIG proteins, nucleotide sequences, probes, andantibodies. In a single sentence in the embodiment, the authorsimplicate that detection of MIG may potentially be used to bioassay forIFN-γ. It is suggested that a sample containing an unknown quantity ofIFN-γ is applied to a macrophage or monocytic cell line and the amountof MIG proteins, or MIG messenger RNA which is made in response to theapplied IFN-γ may be subsequently quantified. Quantification is taughtto be possible by any means known in the art, such as radioimmunoassay,Northern blots, Western blots, enzyme-linked immunoadsorbent assay, etc.The amount of the MIG protein or mRNA produced by the cells in responseto the applied IFN-γ would potentially correlate with the amount ofIFN-γ in the sample (but no evidence is presented). See WO 92/10582 atpp. 8-9. There are no embodiments in the PCT for using MIG as a bioassayfor IFN-γ. More, specifically, there are no embodiments described usingthe detection of MIG as a marker of antigen-specific immuneresponsiveness, or antigen-specific IFN-γ production. Moreover, there isno description of how MIG expression could be used as a marker fordetecting antigen-specific cells or for detecting antigen-specific IFN-γproducing cells. Thus, there is a need in the art for a more sensitiveassay that uses the induction of MIG expression as a marker for antigenspecific IFN-γ producing cells or antigen-specific IFN-γ production.While Farber implicates the use MIG as a bioassay for IFN-γ, ourapplication describes induction of MIG expression as a marker for immuneresponsiveness.

[0016] Amichay et al. 1996 (Genes for chemokines HuMig and Crg-2 areinduced in protozoan and viral infections in response to IFN-γ withpatterns of tissue expression that suggest nonredundant role in vivo. J.Immunol. 157:4511) teach in vivo MIG expression following exposure to apathogen. In that report, mice were experimentally infected withprotozoan or viral pathogens and the level of MIG expression wasassessed. Compared to non-exposed controls, induction of MIG expressionwas noted in various organs and tissues in response to infection,demonstrating that MIG expression is induced following in-vivoinfection. Specifically, Amichay demonstrated that infection of micewith different pathogens induced expression of MIG in various organs.Induction of MIG expression following in vivo infection was not observedif they used IFN-γ knockout mice (mice that are genetically incapable ofproducing IFN-γ). Similarly, injecting mice with IFN-γ also induced MIGexpression in various organs as well. These studies did not demonstratethat induction of MIG expression was antigen-specific orgenetically-restricted or general to inflammatory stimuli induced byinfection, or if it was a consequence of IFN-γ production fromantigen-specific cells or if production was specifically mediated byCD8⁺ T cells, CD4⁺ T cells, and/or NK cells. Accordingly, there is aneed in the art to develop a sensitive and specific assay for IFN-γactivity or other Th−1 cytokine which is based on the detection of MIGor other chemokine as a marker for antigen-specific immuneresponsiveness or for detecting antigen specific IFN-γ cells.

[0017] Recent studies have implicated MIG as an important immuneeffector molecule in its own right. Like IP-10 and I-TAC, MIG binds to acommon receptor, CXCR3, which is known to be expressed on the surface ofactivated/memory T cells and NK cells (28). These chemokines are inducedby a variety of cell types in response to IFN-γ (23,29,30). MIG has beenshown to enhance NK cell mediated cytotoxicity and to mediate antitumorand antiviral responses in vivo (31, 32). Neutralization of MIG has alsobeen shown to prolong graft survival in vivo (33). Since expression ofMIG mRNA can be detected in a variety of different organs followingIFN-γ administration, including liver, thymus, lung and spleen, or inthe liver and spleen of mice following infection by P. yoelli or T.gondii (7), it is likely that MIG may represent a key mediator ofprotective immunity.

SUMMARY OF THE INVENTION

[0018] The evaluation of antigen-specific immune responses is criticalfor understanding the mechanisms of immune protection, for establishingthe efficacy of candidate vaccines, and for diagnostics. Here, wedescribe a sensitive and specific assay for detecting antigen-specificMIG expression and/or antigen-specific IFN-γ activity which is based onthe detection of the chemokine monokine induced by gamma interferon(MIG) as a measure of the biological effect of IFN-γ, rather than directquantitation of IFN-γ or IFN-γ secreting cells per se. In laboratorystudies, upregulation of MIG expression was observed following in vitroactivation of PBMC with defined CD8⁺ T cell epitopes derived frominfluenza virus (FLU), CMV, or EBV, and in all cases induction of MIGexpression was antigen-specific, genetically restricted and dependent onboth CD8⁺ T cells and IFN-γ. Further, antigen-specific MIG expressionwas also demonstrated in an experimental vaccine model using volunteersimmunized against malaria. Responses as assessed by the MIG assayparalleled those detected by conventional IFN-γ ELISPOT, but themagnitude of response and sensitivity of the MIG assay were superior.This was demonstrated by the ability of the MIG assay to detectantigen-specific immune responses that were not detectable as positiveresults using the IFN-γ ELISPOT assay, and by depletion-reconstitutionstudies which demonstrated that the sensitivity of the MIG assay couldbe least twice as sensitive than the ELISPOT assay for detectingpositive responses. Further demonstration of the sensitivity ofdetecting induction of MIG expression compared to IFN-γ responses isalso provided by detection of MIG protein, but not IFN-γ, by ELISA usingcell culture supernatant. Our data validate this novel method for thedetection of low levels of antigen-specific and genetically restrictedIFN-γ activity.

OBJECTS OF THE INVENTION

[0019] Accordingly, an object of this invention is a rapidimmunodiagnostic assay method for detecting antigen-specific MIGexpression or antigen-specific IFN-γ production or IFN-γ producing cellsby detecting MIG expression.

[0020] Another object of this invention is a method of assessing theeffectiveness of a compound or system in inducing an immune response bydetecting the amount of MIG expression.

[0021] Still another object of this invention is the detection of lowlevels of IFN-γ through detecting MIG expression.

[0022] Yet another object of this invention is a method of assessingantigen-specific immune responsiveness through the detection of MIGexpression.

[0023] Another object of this invention is an immunoassay method fordetection of MIG expression during CD8⁺ deletion and add-backexperiments.

[0024] A further object of the invention is an immunoassay kit fordetecting IFN-γ or IFN-γ secreting cells.

[0025] A yet further object of the present invention is the detection ofCD8 IFN-γ-producing cells or CD8⁺ T cell-mediated IFN-γ product.

[0026] A still further object of the present invention is the detectionof CD4 IFN-γ-producing cells.

[0027] A yet additional object of the present invention is the detectionof CD8⁺/CD4⁺ IFN-γ-producing cells.

[0028] A still additional object of the present invention is thedetection of cytokines that upregulate the production of MIG or thedetection of cytokines whose production is upregulated by IFN-γ.

[0029] These and additional objects of the invention are accomplished bya detection method and kit which is based, at least in part, on thebiological amplification of Th−1 cytokine responses, thus providing asensitive means for detecting high as well as low levels ofantigen-specific Th−1 cytokine or antigen-specific Th−1 cytokineproducing cells and a means for assessing the effectiveness of compoundsor systems in inducing an immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] A more complete appreciation of the invention will be readilyobtained by reference to the following Description of the PreferredEmbodiments and the accompanying drawings in which like numerals indifferent figures represent the same structures or elements. Therepresentations in each of the figures is diagrammatic and no attempt ismade to indicate actual scales or precise ratios. Proportionalrelationships are shown as approximations.

[0031]FIG. 1 is a representation of the antigen-specific induction ofMIG expression mediated by IFN-γ.

[0032]FIG. 2 is a representation of an example MIG assay and method.

[0033]FIG. 3 is a graph of the induction of chemokine expression byIFN-γ.

[0034]FIG. 4 is flow cytometry data showing the antigen-specific andgenetically restricted induction of MIG expression.

[0035]FIG. 5 is a bar graph showing that MIG expression is dependent onIFN-γ.

[0036]FIG. 6 is a bar graph showing the kinetics of antigen-specificinduction of MIG expression.

[0037]FIG. 7 is a bar graph showing the implicated cellular mechanism ofantigen-specific induction of MIG expression.

[0038]FIG. 8 is a bar graph showing the effect of selective CD8⁺ T cellenrichment on the antigen-specific induction of MIG expression.

[0039]FIG. 9 is a bar graph showing the antigen-specific induction ofMIG expression in an example vaccine model.

[0040]FIG. 10 is a bar graph that demonstrates the ability to detectantigen-specific induction of MIG expression using whole bloodstimulation protocols.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] This invention is directed to an immunodiagnostic assay andmethod to detect cytokines that upregulate the production of IFN-γ, orto detect cytokines whose production is upregulated by MIG. Typically,the invention includes an immunodiagnostic assay and method to detectMIG or to detect IFN-γ or IFN-γ secreting cells by assessing the amountof MIG expression. The vertebrate immune system is a complex system thatis comprised of various organs, tissues, cells and cells products thatprovide protection against pathogenic organisms, toxins, or neoplasticcells. The immune system can be further divided into the innate immunesystem and the acquired immune system. The focus of this patent will beon the acquired immune system, specifically cellular immune responseswhich are mediated by at least three functionally distinct classes ofcells: CD4⁺T cells, CD8⁺ T cells, and NK cells. Activation of thesecells is mediated in part by the recognition of antigenic peptidefragments that are presented in conjunction with majorhistocompatibility molecules (MHC). Through receptors on the cellsurface, CD4⁺ T cells recognize peptides presented by MHC class IImolecules, while CD8⁺ T cells recognize peptides presented by MHC classI molecules. The MHC complex is considered the most polymorphic regionof the human genome and consists of at least 200-1000 different geneswhich may express 8-10 different alleles. Both MHC class I and class IImolecules contain a groove within their structure which allows forbinding of peptides which fit precisely into a specific MHC haplotype. Tcells and NK cells express a distinct receptor on their cell surfacewhich can recognize a specific antigen/MHC complex. The interaction ofthese cell surface molecules mediates the specificity of the immuneresponse. Thus, a given T cell or NK cell is only activated byrecognition of a specific and distinct antigen/MHC complex, and thebinding of an irrelevant peptide into the MHC binding groove will notactivate those specific cells. Thus, antigen-specific immune responsesrefers to the ability of the immune system to distinguish a specificantigen/MHC complex from one another. The response is said to begenetically-restricted if expression of the antigenic peptide isrestricted to a given MHC haplotype.

[0042] For instance, if a cell in the body were to be infected with avirus, antigens encoded by the virus could be expressed on the surfaceof the infected cells in conjunction with MHC molecules. T cells and NKcells are to recognize the antigen/MHC complex and are activated toeliminate the infection. Elimination of infection could be achieved by avariety of different measures, including lysis of infected cells by CTLsand NK cells or through the production of cytokines which may in turnactivate protective pathways inside the infected cell, or preventinfection from spreading to neighboring cells.

[0043] The invention described herein teaches a new method for detectingthe presence of antigen-specific cytokine (e.g. IFN-γ)-producing cellsthrough the detection of an effect of its activity, the induction of theexpression of a second cytokine, e.g., MIG. As mentioned above,cytokines such as IFN-γ mediates a variety of biological responses, onesuch response is the induction of chemokines, such as MIG. Both MIG andIP-10 are two chemokines belonging to the α superfamily of chemokinesthat are known to be induced following exposure to recombinant IFN-γ.However, it is known that IP-10 can be induced by factors other thanIFN-γ including IFN-α, IFN-β, and LPS while MIG expression is notinduced by these factors. The proposed model for antigen-specificinduction of MIG expression mediated by IFN-γ is shown in FIG. 1. Anantigen-presenting cell (APC) expresses a specific antigen-MHC class Icomplex on its cell surface for recognition by immune cells, namely CD8⁺T cells, or NK cells. This antigen-specific, genetically-restrictedinteraction activates immune cells to produce IFN-γ. Production of IFN-γinduces the expression of MIG in the cognate APC, as well as neighboringAPC. Thus, detecting MIG expression is the result of biologicalamplification of IFN-γ production and provides a specific and sensitivemeans of detecting low levels of IFN-γ activity and IFN-γ producingcells, or other Th-1-type cytokine-producing cells. This is a uniquefeature for detecting antigen-specific IFN-γ producing cells through thedetection of MIG expression by flow cytometry or other means known bythose skilled in the art. Such means include but are not limited to RIA,ELISA, ELISPOT, RT PCR, and flow-based assays, and bead-based assays. Ina flow based assay, the sample is detected (e.g., by fluorescence orother means) while flowing through detection windows. Flow-based assaysinclude, e.g., flow cytometry. In a bead-based assay, a fluorescent beadis covalently attached to a detecting antibody. A bead-based assay canbe analyzed by any flow cytometry or any device or method that canquantitatively detect fluorescent beads. Detection of antigen-specificcells by flow cytometry offers many advantages over more conventionalassays.

[0044] Advantages and New Features

[0045] In addition to sensitivity, the instant assay has a number ofadditional advantages over other assays. For example, antigen-specificIFN-γ restricted responses can be readily detectable by MIG assayfollowing a few hours of incubation with antigen. In the examplesprovided herein, antigen-specific induction of MIG expression wasdetectable following 4-16 hours of culture. In contrast, those of skillin the art frequently utilize longer culture time for the ELISPOT assay(24-36 hours and as long as 13 days). Additionally, even if the variableculture time is discounted from each method the MIG assay is at least2.85 times faster to perform from start to finish than the ELISPOT assaywhich is comparable to other current detection methods. (Table IV).Thus, the MIG assay also provides a method for detecting antigenspecific IFN-γ responses that is less laborious and requires less timeto complete and evaluate.

[0046] Furthermore, because the assay of the present invention can be aflow cytometry-based assay, it can be technically simple and arelatively rapid and objective method for identifying antigen-specificcells. However, current protocols for detecting antigen-specificcytokine producing cells by intracellular staining and flow cytometry donot provide a robust means of reproducibly detecting low levels ofantigen-specific, cytokine producing cells at significant levels abovebackground. Because antigen-specific induction of MIG expression is theresult of biological amplification of IFN-γ responses, it provides anovel alternative for detecting low levels of antigen-specific, IFN-γrestricted responses by flow cytometry and a measure of immuneresponsiveness. Detection of MIG expression by flow cytometry evaluatesthe frequency of responsive cells and magnitude of response at alarge-scale, single cell level, as compared with the ELISPOT assay whereit is the number of responsive cells in a limited sample in each wellthat are detected and enumerated. Antigen-specific detection of MIGexpression by flow cytometry is more sensitive than the IFN-γ ELISPOTassay because it is based on the biological amplification of IFN-γresponses, allowing the detection of positive responses that are notscored positive by the IFN-γ ELISPOT assay conducted in parallel. Asshown in the Examples disclosed herein, the use of CD8⁺ T cell depletionand add-back experiments provides one example demonstrating that the MIGassay can be at least two times more sensitive than the IFN-γ ELISPOTassay.

[0047] In a typical embodiment, the invention uses a new assay fordetecting IFN-γ other Th−1 cytokines or cells secreting IFN-γ or otherTh−1 cytokines by measuring one of the biological effects of theactivity of the cytokine through flow cytometric detection of thechemokine whose production is upregulates. The detection of IFN-γ (orother Th−1 cytokine) or IFN-γ producing (or other Th−1 producing) cellscan be done using samples obtained from a variety of bodily fluids,tissues, or cells, including whole blood, plasma, serum, PBMCpreparations, saliva, tears, and biopsy samples.

[0048] We describe a method for detecting MIG (or a cytokine whoseproduction is upregulated by a Th−1 cytokine) or a Th−1 cytokine (e.g.,IFN-γ) or Th−1 cytokine (e.g., IFN-γ) producing cells using peripheralblood mononuclear cells (PBMC's) isolated from whole blood. The generalprinciple of the assay is as follows. A sample is obtained from a hostand cultured with an antigen, such as a known pathogen or fragmentthereof. Culture of the sample with an antigen serves to activate immunecells to produce the Th−1 cytokine, e.g. IFN-γ. The Th−1 cytokine inturn induces the production of chemokine (e.g., MIG) molecules which aredetected by flow cytometry. (FIG. 2). Alternative means for detectingthe level of chemokine expression can include, but are not limited to,ELISPOT, RIA RT-PCR, bead-based assays, etc. Detection of chemokinemolecules for the purpose of this invention can include MIG DNA, RNA,full-length protein or a peptide thereof.

[0049] In another embodiment, the detection of chemokine (e.g., MIG)expression from ex vivo samples can be used to determine if a compoundor system is effective in inducing IFN-γ (or other Th−1 cytokine)responses in individuals. Compounds include substances known to enhanceimmune responses such as adjuvants or systems such as whole organisms,subunits of whole organisms, or vaccine delivery systems. Baselinelevels of chemokine (e.g., MIG) expression could be determined in theindividual prior to immunization using compounds or novel systems.Following immunization, a sample from the individual could be obtainedand chemokine expression detected and compared to baseline levels ofthat chemokine's expression or to control individuals who were notimmunized. Both vaccinated and unvaccinated individuals could also beadministered a fragment of the vaccine, and samples obtained from theseindividuals could be used to assess the level of antigen-specificinduction of chemokine expression in immunized and non-immunizedindividuals as a measure of immune responsiveness.

[0050] Additionally, the method can be used to determine the immunestatus of a mammal, such as a human. Various diseases, such as HIV, andgenetic conditions, reduce the immunoresponsiveness of their patients byattacking cells that mediate the expression of MIG or similarcytokine/chemokines whose production is upregulated by IFN-γ or otherTh−1 cytokines. Thus, an assay for MIG (or other chemokine) expressioncan effectively monitor the progress of the disease of the response of asubject to treatment.

[0051] In another embodiment, the method could be incorporated into akit, which may or may not include an antigen, such as a known pathogenor fragment thereof, and other reagents, such as indictors for MIG orfor other chemokines whose expression is upregulated by IFN-γ or otherTh−1 cytokines. Indicators for MIG and similar chemokines aresubstances, such as labeled (e.g., fluorescently) antibodies to MIG,that allow the specific detection and quantitation of MIG or the desiredchemokine. Typically, indicators for MIG or similar chemokines bindcovalently to MIG or the desired chemokine, either by direct attachmentor via an intermediate molecule. The kit and assay could be performed byobtaining a sample in a field or laboratory or clinical setting, and theamount of MIG or similar chemokine expression could be determined byvarious means, e.g., flow cytometry, RIA, ELISPOT, ELISA, RT-PCR, orbead-based assays.

[0052] Although the invention has been discussed mainly with respect toMIG and IFN-γ, those skilled in the art will recognize that thedetection of MIG expression may be used as a marker for the presence andquantitation of other cytokines whose production it upregulates.Similarly, other cytokines that upregulate the production of IFN-γ orother Th−1 cytokines may be used as a marker for the presence andquantitation of IFN-γ or other cytokines.

[0053] Having described the invention, the following examples are givento illustrate specific applications of the invention, including the bestmode now known to perform the invention.

EXAMPLE 1

[0054] PBMC samples and cell culture

[0055] Study subjects were healthy Caucasian volunteers, aged 22-51, whowere seronegative for HIV gp120 antibodies and HBV core antibodies, asdetermined by standard clinical screening. HLA allelic frequencies wereestablished from peripheral blood samples using standard site-specificoligonucleotide PCR typing). PBMC were isolated by standard gradientcentrifugation over Ficoll-Paque (Amersham Pharmacia Biotec AB, Uppsala,Sweden). Cells were cultured in RPMI 1640 containing 10 mM Hepes andsupplemented with 10% heat-inactivated FCS (Sigma Chemical Co., St.Louis, Mo.), 2 mM L-glutamine, 100U/ml penicillin, and 100 ug/mlstreptomycin (Life Technologies, Grand Island, N.Y.). In someexperiments, PBMC were depleted of specific subsets (CD4⁺, CD8⁺, CD16⁺or CD56⁺ cells) prior to culture using MACS beads (Miltenyi Biotec,Auburn, Calif.), as described by the manufacturer. Depleted cultureswere analyzed by flow cytometry and in all cases the efficiency ofdepletion was at least 95%. All experiments reported herein wereconducted using fresh PBMC. Other studies have established that similarresponses can be detected using frozen PBMC (data not shown).

[0056] Synthetic peptides

[0057] Synthetic 9-mer or 10-mer peptides representing wellcharacterized HLA-A*0201 restricted epitopes from the influenza matrixprotein (FLU, residues 58-66) (9), cytomegalovirus phosphoprotein (CMV,residues 495-503, (10, 11), human immunodeficiency virus gag protein(HIV, residues 75-85) (9), and hepatitis B virus core antigen (HBV,residues 18-27) (12) were purchased from Chiron Corporation (Clayton,VIC, Australia) or Research Genetics (Huntsville, Ala.) and werepurified to >95%. Peptides representing HLA-DR restricted CD4⁺ T cellepitopes and nested HLA-A*0201 restricted CD8⁺ T cell epitopes from theP. falciparum circumsporozoite protein (CSP) (13-19) were obtained fromChiron Corporation. The 9-mer peptide representing the HLA-B8 restrictedepitope from the Epstein-Barr virus nuclear antigen 3 (EBV, residues339-347) (20). Peptide sequences are presented in Table I.

EXAMPLE 2

[0058] MIG is induced in response to the expression of IFN-γ

[0059] To investigate the profile of chemokines induced followingexposure to IFN-γ which could be readily detected by intracellularstaining and flow cytometry, PBMC were cultured with recombinant humanIFN-γ (rec.hIFN-γ) and stained intracellularly with mAbs to MIG, IP-10,MCP−1, RANTES, or MIP-α. Expression of both MIG and of IP-10 was readilydetectable in PBMC following overnight culture (FIG. 3A). MCP−1 andMIP−1α expression were slightly upregulated compared to media controlbut there was no change in expression of RANTES (data not shown).Additional dose titration experiments demonstrated that MIG expressionwas a more sensitive measure of IFN-γ as compared to IP-10 (FIG. 3B).Moreover, it has been established that the induction of MIG isrestricted to IFN-γ, whereas IP-10 can be induced by factors other thanIFN-γ including IFN-α, IFN-β, and LPS (22-24). Therefore, expression ofMIG, but not IP-10, can be considered a surrogate marker of IFN-γspecific activity, and subsequent studies focused on the induction MIGexpression.

EXAMPLE 3

[0060] Induction of MIG expression is antigen-specific and geneticallyrestricted We evaluated whether MIG expression could be induced in anantigen-specific and genetically restricted manner. PMBC from volunteersknown to express HLA-A*0201 were cultured with peptides containingHLA-A*0201-binding CD8⁺ T cell epitopes derived from HIV, HBV, FLU andCMV. Representative flow cytometric data from one experiment ispresented in FIG. 4. MIG expression was detected with PBMC fromHLA-A*0201-positive volunteers (Vols. #1,2 and 3) activated with CMV andFLU peptides, but not HIV or HBV peptides indicating that the responsewas antigen-specific. Additionally, antigen-specific induction of MIGexpression was not detected in cultures from volunteers who did notexpress the HLA-A*0201 allele (Vols. #15 and #16) demonstrating that theantigen-specific response was genetically restricted. These dataestablished that culture of PBMC with synthetic peptides elicitedantigen-specific and genetically-restricted MIG expression,

EXAMPLE 4

[0061] Induction of MIG expression is IFN-γ dependent

[0062] Having established that rec.hIFN-γ induced MIG expression andthat culture of PBMC with synthetic peptides elicited antigen-specificand genetically-restricted MIG expression, we determined whether theinduction of MIG expression was dependent on IFN-γ. Accordingly, PBMCwere cultured with FLU or CMV peptide in the presence or absence ofneutralizing mAbs to IFN-γ or a control mAb (mIgG). Analysis of meansand standard deviation of quadruplicate culture and staining wells isshown in FIG. 5. In two volunteers, the addition of neutralizing mAbs toIFN-γ inhibited antigen-specific induction of MIG expression by anaverage of 87% and 85% to the FLU and CMV peptides, respectively. Inrepeated experiments with PBMC from volunteers 1-3, the addition ofneutralizing antibodies to IFN-γ inhibited antigen-specific induction ofMIG expression to both FLU and CMV peptides by 86%, indicating thatantigen-specific induction of MIG expression was dependent upon IFN-γ.

EXAMPLE 5

[0063] Kinetics of antigen-specific induction of MIG expression

[0064] Since IFN-γ is rapidly produced by T cells following exposure toantigen, we evaluated the kinetics of antigen-specific induction of MIGexpression. Antigen-specific induction of MIG expression was found to berapidly induced in PBMC cultured with CMV and EBV peptides and wasreadily detectable after 4 hours of culture (FIG. 6A and B). Theseresults demonstrate that the antigen-specific induction of MIGexpression is rapid (within 4 hours) and is sustained for at least 16hours, at least for these peptides. Early kinetic experiments were alsoconducted with the FLU peptide (FIG. 6C). With this peptide,antigen-specific induction of MIG expression was detected in one of twovolunteer after 4 hours of culture (Vol.#5), but increased withadditional time and was readily detectable in both volunteers following6 hours of culture (FIG. 6C). In these cultures, the rapid induction ofMIG expression was shown to be dependent upon IFN-γ from activatedcells, as the addition of neutralizing antibodies to IFN-γ inhibited theantigen-specific induction of MIG expression. Antigen-specific IFN-γresponses could also be detected by ELISPOT at similar time points (datanot shown), and studies are underway to more precisely define the earlykinetics of response for both MIG and ELISPOT assays.

EXAMPLE 6

[0065] Cellular requirements for optimal antigen-specific induction ofMIG expression

[0066] The HIV, HBV, CMV, FLU and EBV peptides used in these studieswere 9-mers or 10-mers known to be restricted by the HLA-A*0201 orHLA-B8 Class I molecules. Therefore, we reasoned that theantigen-specific, MHC-restricted induction of MIG expression wasmediated by CD8⁺ T cells following recognition of the peptide/MHCcomplex on the surface of the APC. To test this, PBMC were depleted ofCD8⁺ T cells (CD8⁻). In all cases, depletion of CD8⁺ T cellssignificantly inhibited the antigen-specific induction of MIGexpression, demonstrating that CD8⁺ T cells were mediating theantigen-specific response. The analysis with means and standarddeviation of quadruplicate wells is presented in FIG. 7. The dependenceon CD8⁺ T cells for MIG expression was confirmed in subsequent selectiveenrichment studies where the induction of MIG expression from CD8⁺ Tcell depleted cultures could be reconstituted by the addition of CD8⁺ Tcells.

[0067] Both CD4⁺ T cells and NK cells are known to be major producers ofIFN-γ (1). In addition, CD56⁺ and CD16⁺ are considered prototypicmarkers for NK cells, although a recent study suggests that CD56 mayalso represent a marker for CD8⁺ effector T cells (25). Accordingly, toinvestigate the role for these cells in the induction of MIG expression,we specifically depleted PBMC cultures of CD4⁺, CD56⁺ or CD16⁺ cells. Asshown in FIG. 7 depletion of CD4⁺ T cells or CD56⁺ cells (or CD16⁺cells; data not shown) did not inhibit the robust expression of MIG incultures activated with either the CMV or EBV peptides. However,depletion of CD4⁺ T cells or CD56⁺ cells (or CD 16⁺ cells; data notshown) did decrease the more modest induction of MIG expression incultures activated with the FLU peptide.

[0068] Requirements for IL-12 and IL-2 for bystander induction of MIGexpression Both IL-2 and IL-12 are known to induce IFN-γ production byCD4⁺ T cells and NK cells (26). Therefore, to investigate if IL-2 andIL-12 were involved in bystander activation, neutralizing mAbs to IL-12or to CD122 were added to cultures activated with the FLU, CMV or EBVpeptides. CD122 (IL-2Rα) is part of the IL-2 receptor complex andantibodies to this component are known to block the binding of IL-2(27). As shown in FIG. 7, addition of neutralizing mAbs against IL-12 orIL-2 had no effect on the response to either the CMV or EBV peptides.However, addition of mAbs to IL-12 and CD122 did decrease MIG expressionin PBMC cultured with the FLU peptide. These results are consistent withobservations from the depletion experiments described above, and suggestthat the CD4⁺ T cells and NK cells required for optimal induction of MIGexpression in the FLU system are activated nonspecifically through IL-12and IL-2 cytokine feedback loops. In contrast, antigen-specificinduction of MIG expression to CMV or EBV peptides appears to bedependent on CD8⁺ T cells but not dependent on bystander activation ofCD4⁺ T cells or NK cells for optimal expression. Furthermore, therequirement for bystander activation appears to be reflected in themagnitude of the antigen-specific MIG response, since a more robustresponse was observed with the CMV and EBV peptides as compared with theFLU peptide. The magnitude of the antigen-specific induction of MIGexpression directly correlated with the frequency of antigen-specificIFN-γ-producing cells as determined by parallel IFN-γ ELISPOT, as shownin Table II.

EXAMPLE 7

[0069] Comparison of MIG, MIG-ELISA, and ELISPOT assays MIG assay

[0070] PBMC were cultured at a concentration of 0.5×10⁶ cells/well in atotal volume of 200 μl complete medium in a 96-well round bottom plateat 37° C., in an atmosphere of 5% CO2. Synthetic peptides were added ata final concentration of 10 μg/ml prior to initiation of culture.Brefeldin-A or monesin were not added to cultures. Unless otherwiseindicated, effectors for the MIG assay were cultured overnight (16-20hours), in triplicate or quadruplicate. Then, PBMC were washed once incold Dulbecco's PBS, and stained with mAbs to CD14 or CD11a (BectonDickinson, San Jose Calif.). PBMC were permeabilized withCytofix/Cytoperm (Pharmingen, San Diego, Calif.) according tomanufacturer's instructions, and stained intracellularly withPE-conjugated mAbs to the human chemokines MIG, IP-10, MCP-1, MIP-1α,RANTES, or IFN-γ (Pharmingen) at a concentration of 0.4 μg antibody/10⁶cells. Samples were acquired on a Becton Dickinson FACSCAN (San Jose,Calif.). For each analysis, at least 25,000 events were acquired andcells were gated within the monocyte/macrophage population based uponforward scatter (FSC) and side scatter (SSC) characteristics. Gatedcells were analyzed for percentage of CD14- or CD11a-positive cellscounter-stained with anti-MIG mAb using the CellQuest software (BectonDickinson).

[0071] IFN-γELISPOT assay

[0072] The number of peptide-specific IFN-γ producing cells wasdetermined by ELISPOT assay, basically as described elsewhere (21). Inbrief, sterile 96-well multiScreen-IP MAIP plates (Millipore, Bedford,Mass.) were coated overnight at 4° C. with 50 μl of PBS containing 10μg/ml of anti-IFN-γ mAb (clone 1-D1K; Mabtech, Stockholm, Sweden). Wellswere washed 6 times with RPMI-1640 and blocked for 1 hour at roomtemperature with 100 μl of RPMI-1640 supplemented with 10% FCS. Then,100 μl of input PBMC (5×10⁵−2.5×10⁵ PBMC) were added in quadruplicate,together with 100 μl of test or control peptide at a final concentrationof 10 μg/ml. Unless otherwise indicated, cultures were incubated for 36hours at 37° C. in an atmosphere of 5% CO₂. Wells were then washed 6times with PBS/0.05% Tween 20 (Sigma chemical Co., St. Louis, Mo.), andincubated for 3 hours at room temperature with 100 μl of 1 μg/mlbiotinylated anti-IFN-γ mAb (clone 7B6-1, Mabtech). Wells were againwashed 6 times with PBS/0.05% Tween 20 and incubated for 1 hour at roomtemperature with 100 μl of 1:1000 dilution of streptavidin alkalinephosphatase (Mabtech). Wells were washed 6 times with PBS/0.05% Tween 20and 3 times with PBS, and then developed with 100 μl of 1:25 dilutedalkaline phosphatase substrate (Bio-Rad, Hercules, Calif.). Thecolorimetric reaction was stopped after 15 minutes by extensive washingin tap water and plates were air-dried. The number of spotscorresponding to IFN-γ-producing cells in wells (IFN-γ spot formingcells; SFCs) were enumerated with the Zeiss KS ELISPOT system (CarlZeiss Inc., Thornwood, N.Y.). Responses were expressed as the number ofIFN-γ secreting cells (spot-forming colonies, SFCs) per 10⁶ PBMC.

[0073] MIG ELISA

[0074] Immunolon 2HB 96-well plates (Dynex Technologies, Chantilly, Va.)were coated overnight at 4° C. with 100 μl of PBS containing 2.5 μg/mlof anti-MIG mAb (clone B8-11; Pharmingen). Plates were washed 3 timeswith PBS/0.05% Tween 20 and blocked for 2 hours at room temperature with100 μl of PBS supplemented with 1% FCS. Test samples of cell culturesupernatant and recombinant human MIG standards (Pharmingen) were thenadded in 100 μl volumes in duplicate, and cultures were incubated for 2hours at room temperature. After 3 washes, 100 μl of PBS containing 2μg/ml of biotinylated anti-MIG mAb (clone B8-6; Pharmingen) was added toeach well, and cultures were again incubated for 2 hours at roomtemperature. Plates were then washed 3 times and 100 μl ofHRP-Streptavidin conjugate (1:2000 dilution; Zymed Laboratories, SanFrancisco, Calif.) was added for 1 hour at room temperature. Plates werewashed 3 times and developed with ABTS substrate solution (KPL,Gaithersburg, Md.) according to the manufacturer's protocol.Concentrations were calculated by interpolation from standard curvesbased on recombinant cytokine dilutions run in parallel on the sameplate.

[0075] IFN-γELISA

[0076] Human IFN-γ ELISA kits were purchased from Endogen (Woburn,Mass.) and used according to manufacturer's instructions. Concentrationswere calculated as described above for the MIG ELISA assay. Thesensitivity of this IFN-γ ELISA was 2 μg/ml.

[0077] Neutralizing antibody treatments

[0078] Neutralizing mAbs to IFN-γ (clone B27) and IL-12 (clone C8.6),and blocking antibody to CD122 (clone Mik-γ2) or control mAb (cloneMOPC-21), were purchased from Pharmingen. In all studies reportedherein, mAbs were added at a final concentration of 20 μg/ml at theinitiation of culture and maintained throughout the culture period.

[0079] Statistical Analysis

[0080] The significance of group differences for the MIG and ELISPOTassays was calculated using the Student's t-Test (Microsoft ExcelVersion 8.0, Microsoft Corporation). Responses were considered positiveif the response to test peptide (FLU, CMV, EBV, or CSP) wassignificantly different (p<0.05) as compared with the response tonegative control peptides (HIV or HBV) and if the stimulation index(SI=response with test peptide/response with control peptide) wasgreater than 2.0.

[0081] Comparisons

[0082] ELISPOT assays are now routinely used to detect and quantitateantigen-specific cytokine-secreting cells. Since our assay for MIGexpression represents an indirect measure of IFN-γ production fromantigen-specific cells following biological amplification, we nextdirectly compared the MIG and ELISPOT assays in parallel with samplesfrom the same volunteer using the same peptide. These experiments aresummarized in Table II. In multiple parallel studies, the level of MIGexpression directly correlated with the number of IFN-γ SFCs obtained byELISPOT (R²=0.94), supporting our hypothesis that MIG represents asurrogate marker for antigen-specific, IFN-γ-producing cells. In allinstances where the ELISPOT assay was considered positive, thecorresponding sample met the criteria for positivity in the MIG assay.Moreover, in all instances where cultures were considered positive byeither assay, a higher stimulation index was noted with the MIG assay ascompared with the ELISPOT assay (average at least 3-fold higher).Finally, in three instances (Vols. #3, 4 and 5 in response to FLU), theresponse as assessed by the MIG assay was significant, but thecorresponding response as measured by ELISPOT was not significant.

[0083] We cannot definitively exclude the possibility that these latterresponses as assessed by the MIG assay represented false positives.However, in both the ELISPOT and the MIG assays, responses wereconsidered positive only if the response to test peptide wassignificantly different from the response to negative control peptide(s)and if the stimulation index was 2.0. Although the response to the FLUpeptide as compared with the negative control peptides in volunteer #4was significant, the stimulation index was only 1.7, and this responsedid not meet our criteria for positivity. In addition, in repeatedexperiments using PBMC volunteers #3, 4 and 5, positive responses wereconsistently detected with the MIG assay. These data indicate that thepositive responses detected by MIG but not ELISPOT assay do notrepresent false positives. These data further demonstrate the enhancedsensitivity of the MIG assay for detecting antigen-specific, IFN-γdependent immune responses.

EXAMPLE 8

[0084] Enhanced Sensitivity of the MIG assay, as compared with otherassays, for detecting IFN-γ-mediated antigen specific immune responses

[0085] To further demonstrate the sensitivity of the MIG assay, and toexclude the possibility that the assay was detecting false positives, wedirectly compared the MIG and ELISPOT assays in selective enrichmentstudies using PBMC from volunteers known to respond to a given peptide.For these studies, PBMC from known responders were depleted of CD8⁺ Tcells (<1% and <2% CD8⁺ T cells for Vols. #2 and #10, respectively) anddefined numbers of CD8⁺ T cells were added back to the CD8⁺ depletedsamples and cultured with either FLU or EBV peptides for 16 hours. Inall cases, when compared to HIV or media controls, significant responses(black bars) were detected by the MIG assay before they were detected bythe ELISPOT assay conducted in parallel (FIG. 8). In volunteer #2, whosePBMC contained 22% CD8⁺ T cells, positive responses were detected byELISPOT when 4% CD8⁺ T cells were added back to the depleted culture. Inthe MIG assay conducted in parallel with the same sample, however,responses classified as positive could be detected when only 2% CD8⁺ Tcells were added back to depleted cultures. In volunteer #10 whose PBMCcontained 10% CD8⁺ T cells, positive responses were detectable by theELISPOT when 4% CD8⁺ T cells were added back, and by the MIG assay whenonly 1% CD8⁺ T cells were added back to depleted cultures. Thus, forthese volunteers, the MIG assay was 2 to 4 fold more sensitive than theELISPOT assay for detecting antigen-specific immune responses.

[0086] Antigen-specific induction of both MIG and IFN-γ were alsoevaluated by cytokine specific ELISAs. Cell-free culture supernatantsobtained from samples used in the kinetic studies (FIG. 6) were takenfollowing 8 and 16 hours of culture and evaluated for MIG and IFN-γ byELISA. As summarized in Table III, MIG could not be detected in samplescollected following 8 hours of culture, but was readily detectable inculture supernatant following 16 hours of culture with either the CMVand EBV peptides. As detailed earlier, antigen-specific induction of MIGexpression could be detected by flow cytometry within 4 hours ofculture, highlighting the sensitivity of the flow-based technique (FIG.6). IFN-γ was not detectable in culture supernatants from either timepoint.

[0087] In summary, these data establish that the MIG assay provides aspecific and sensitive means of detecting low levels of IFN-γ activityand further demonstrate that this assay can detect responses that arebelow the level of sensitivity of both the standard IFN-γ ELISPOT assayand a MIG-based ELISA assay.

EXAMPLE 9

[0088] Induction of antigen-specific MIG expression in a vaccine model(volunteers immunized with malaria sporozoites)

[0089] Therefore, we have established that the MIG assay could alsodetect responses in a non-viral infectious disease model (All studiesreported above were conducted with immunogenic virally derived peptideswith PBMC from volunteers naturally exposed to FLU, EBV, or CMVviruses). PBMC from HLA-A*0201-positive volunteers immunized withirradiated Plasmodium falciparum sporozoites were cultured with peptidesderived from the P. falciparum circumsporozoite antigen (CSP). PBMC froman HLA-A*0201 volunteer who was mock-immunized with noninfectedmosquitoes were also cultured with peptides as a control. As shown inFIG. 9, although responses could not be detected to the short malariapeptides (CSP201, CSP202 and CSP203), antigen-specific induction of MIGexpression was observed in both immunized volunteers following culturewith the longer malaria peptide CSP238. Responses to the second peptide,CSP239, were also detected in one of the two volunteers.Antigen-specific responses to malaria peptides could not be detected inPBMC from the mock-immunized volunteer. These results demonstrate thatthe MIG assay is able to detect antigen-specific responses induced byimmunization. TABLE IV Experimental Design of MIG assay compared toELISPOT Assay MIG ELISPOT Coat plates with 2 hours-16 hrs antibodiesPlate samples 1 hr Wash plate 9× .32 hr with antigen Incubate variableBlock plates 1 hr samples Centrifuge .16 hr Plate samples 1 hr Stain .32hr Incubate samples variable Wash 2× .32 hr Wash 6× .32 hr Fix/Perm .32hr Add biotin 4 hrs Wash 2× .32 hr Wash 9× .32 hr Stain .32 hr AddStrep/Avidin 1 hr Wash 3× .32 hr Wash 9× .32 hr Add Developing solution.5 hr Total Time: 3.08 hrs 8.78-24.78 hrs

EXAMPLE 10

[0090] Induction of antigen-specific MIG expression using whole bloodstimulation protocols.

[0091] The previous examples have demonstrated the ability to detectantigen-specific induction of MIG expression using PBMC isolated fromwhole blood by gradient centrifugation. This process is both laboriousand time consuming, and probably involves loss of cells during theprocessing of the samples. As shown in FIG. 10, antigen-specificinduction of MIG expression was readily detectable when whole bloodsamples were incubated with the listed peptides, demonstrating thefeasibility of using unprocessed blood samples for detectingantigen-specific IGN-γ-mediated immune responses. These results alsodemonstrate the frequency of IFN-γ producing cells determined inparallel samples and highlight the potential for a higher magnitude ofresponse as determined by detecting MIG due to the biologicalamplification of the IFN-γ response.

[0092] Whole blood stimulation protocols.

[0093] Peripheral blood samples were collected into heparinizedvacutainer tubes (Becton & Dickinson, San Diego, Calif.) and 1 ml ofblood was aliquoted into round bottom, 15 ml polypropylene tubes.Peptides ( 20 μg/ml were added to each culture and samples wereincubated for 6 hours. After incubation of whole blood with peptides,samples were treated with 2 mM of EDTA for 15 min at room temperature.Erythrocytes were lysed and leukocytes fixed for 10 min at roomtemperature by adding 10 mls of FACS Lysing solution (Becton &Dickinson). Tubes were washed twice and then cells were permeabilizedwith FACES Permabilization solution (Becton & Dickinson) prior tostaining with antibodies to CD14 and MIG.

[0094] REFERENCES

[0095] 1. Boehm, U., T. Klamp, M. Groot, and J. C. Howard. 1997.Cellular responses to interferongamma. Annu Rev Immunol 15:749.

[0096] 2. Doolan, D. L., and S. L. Hoffman. 1999. IL-12 and NK cells arerequired for antigen-specific adaptive immunity against malariainitiated by CD8⁺ T cells in the Plasmodium yoelii model. J. Immunol.163:884.

[0097] 3. Good, M. F., and D. L. Doolan. 1999. Immune effectormechanisms in malaria. Curr Opin Immunol 11:412.

[0098] 4. Sher, A., and R. L. Coffinan. 1992. Regulation of immunity toparasites by T cells and T cell-derived cytokines. Annu Rev Immunol10:385.

[0099] 5. Jouanguy, E., R. Doffinger, S. Dupuis, A. Pallier, F. Altare,and J. L. Casanova. 1999. IL-12 and IFN-gamma in host defense againstmycobacteria and salmonella in mice and men. Curr Opin Immunol 11:346.

[0100] 6. Baggiolini, M., B. Dewald, and B. Moser. 1997. Humanchemokines: an update. Annu Rev Immunol 15:675.

[0101] 7. Amichay, D., R. T. Gazzinelli, G. Karupiah, T. R. Moench, A.Sher, and J. M. Farber. 1996. Genes for chemokines HuMIG and Crg-2 areinduced in protozoan and viral infections in response to IFN-gamma withpatterns of tissue expression that suggest nonredundant roles in vivo. JImmunol 157:4511.

[0102] 8. Schrum, S., P. Probst, B. Fleischer, and P. F. Zipfel. 1996.Synthesis of the CC-chemokines MIP-1alpha, MIP-1beta, and RANTES isassociated with a type 1 immune response. J Immunol 157:3598.

[0103] 9. Parker, K. C., M. A. Bednarek, L. K. Hull, U. Utz, B.Cunningham, H. J. Zweerink, W. E. Biddison, and J. E. Coligan. 1992.Sequence motifs important for peptide binding to the human MHC class Imolecule, HLA-A2. J Immunol 149:3580.

[0104] 10. Parker, K. C., M. A. Bednarek, and J. E. Coligan. 1994.Scheme for ranking potential ULA-A2 binding peptides based onindependent binding of individual peptide side-chains. J Immunol152:163.

[0105] 11. Kern, F., I. P. Surel, C. Brock, B. Freistedt, H. Radtke, A.Scheffold, R. Blasczyk, P. Reinke, J. Schneider-Mergener, A. Radbruch,P. Walden, and H. D. Volk. 1998. T-cell epitope mapping by flowcytometry. Nat Med 4:975.

[0106] 12. Bertoletti, A., F. V. Chisari, A. Penna, S. Guilhot, L.Galati, G. Missale, P. Fowler, H. J. Schlicht, A. Vitiello, R. C.Chesnut, and et al. 1993. Definition of a minimal optimal cytotoxicT-cell epitope within the hepatitis B virus nucleocapsid protein. JVirol 67:2376.

[0107] 13. Calvo-Calle, J. M., J. Hammer, F. Sinigaglia, P. Clavijo, Z.R. Moya-Castro, and E. H. Nardin. 1997. Binding of malaria T cellepitopes to DR and DQ molecules in vitro correlates with immunogenicityin vivo: identification of a universal T cell epitope in the Plasmodiumfalciparum circumsporozoite protein. J Immunol 159:1362.

[0108] 14. Doolan, D. L., S. L. Hoffman, S. Southwood, P. A. Wentworth,J. Sidney, R. W. Chesnut, E. Keogh, E. Appella, T. B. Nutman, A. A. Lal,D. M. Gordon, A. Oloo, and A. Sette. 1997. Degenerate cytotoxic T cellepitopes from P. falciparum restricted by multiple HLA-A and HLA-Bsupertype alleles. Immunity 7:97.

[0109] 15. Good, M. F., D. Pombo, I. A. Quakyi, E. M. Riley, R. A.Houghten, A. Menon, D. W. Alling, J. A. Berzofsky, and L. H. Miller.1987. Human T-cell recognition of the circumsporozoite protein ofPlasmodium falciparum: Immunodominant T-cell domains map to thepolymorphic regions of the molecule. Proc. Natl. Acad. Sci. USA 85:1199.

[0110] 16. Moreno, A., P. Clavijo, R. Edelman, J. Davis, M. Sztein, F.Sinigaglia, and E. Nardin. 1993. CD4⁺ T cell clones obtained fromPlasmodium falciparum sporozoite-immunized volunteers recognizepolymorphic sequences of the circumsporozoite protein. J. Immunol.151:489.

[0111] 17. Moreno, A., P. Clavijo, R. Edelman, J. Davis, M. Sztein, D.Herrington, and E. Nardin. 1991. Cytotoxic CD4⁺ T cells from asporozoite-immunized volunteer recognize the Plasmodium falciparum CSprotein. Int.Immunol. 3:997.

[0112] 18. Zevering, Y., R. A. Houghten, I. H. Frazer, and M. F. Good.1990. Major population differences in T cell response to a malariasporozoite vaccine candidate. Int. Immunol. 2:945.

[0113] 19. Wang, R., D. L. Doolan, T. P. Le, R. C. Hedstrom, K. M.Coonan, Y. Charoenvit, T. R. Jones, P. Hobart, M. Margalith, J. Ng, W.R. Weiss, M. Sedegah, C. de Taisne, J. A. Norman, and S. L. Hoffman.1998. Induction of antigen-specific cytotoxic T lymphocytes in humans bya malaria DNA vaccine. Science 282:476.

[0114] 20. Burrows, S. R., J. Gardner, R. Khanna, T. Steward, D. J.Moss, S. Rodda, and A. Suhrbier. 1994. Five new cytotoxic T cellepitopes identified within Epstein-Barr virus nuclear antigen 3. J GenVirol 75:2489.

[0115] 21. Lalvani, A., R. Brookes, S. Hambleton, W. J. Britton, A. V.Hill, and A. J. McMichael. 1997. Rapid effector function in CD8⁺ memoryT cells. J Exp Med 186:859.

[0116] 22. Farber, J. M. 1990. A macrophage mRNA selectively induced bygamma-interferon encodes a member of the platelet factor 4 family ofcytokines. Proc Natl Acad Sci U S A 87:5238.

[0117] 23. Farber, J. M. 1992. A collection of mRNA species that areinducible in the RAW 264.7 mouse macrophage cell line by gammainterferon and other agents. Mol Cell Biol 12:1535.

[0118] 24. Vanguri, P., and J. M. Farber. 1990. Identification of CRG-2.An interferon-inducible mRNA predicted to encode a murine monokine. JBiol Chem 265:15049.

[0119] 25. Pittet, M. J., D. E. Speiser, D. Valmori, J. C. Ceroftini,and P. Romero. 2000. Cutting edge: cytolytic effector function in humancirculating CD8⁺ T cells closely correlates with CD56 surfaceexpression. J Immunol 164:1148.

[0120] 26. Kobayashi, M., L. Fitz, M. Ryan, R. M. Hewick, S. C. Clark,S. Chan, R. Loudon, F. Sherman, B. Perussia, and G. Trinchieri. 1989.Identification and purification of natural killer cell stimulatoryfactor (NKSF), a cytokine with multiple biologic effects on humanlymphocytes. J Exp Med 170:827.

[0121] 27. Tsudo, M., F. Kitamura, and M. Miyasaka. 1989.Characterization of the interleukin 2 receptor beta chain using threedistinct monoclonal antibodies. Proc Natl Acad Sci USA 86:1982.

[0122] 28. Loetscher, M., B. Gerber, P. Loetscher, S. A. Jones, L.Piali, I. Clark-Lewis, M. Baggiolini, and B. Moser. 1996. Chemokinereceptor specific for IP10 and mig: structure, function, and expressionin activated T-lymphocytes [see comments]. J Exp Med 184:963.

[0123] 29. Farber, J. M. 1993. HuMIG: a new human member of thechemokine family of cytokines. Biochem Biophys Res Commun 192:223.

[0124] 30. Luster, A. D., J. C. Unkeless, and J. V. Ravetch. 1985.Gamma-interferon transcriptionally regulates an early-response genecontaining homology to platelet proteins. Nature 315:672.

[0125] 31. Mahalingam, S., J. M. Farber, and G. Karupiah. 1999. Theinterferon-inducible chemokines MuMIG and Crg-2 exhibit antiviralactivity in vivo. J Virol 73:1479.

[0126] 32. Kanegane, C., C. Sgadari, H. Kanegane, J. Teruya-Feldstein,L. Yao, G. Gupta, J. M. Farber, F. Liao, L. Liu, and G. Tosato. 1998.Contribution of the CXC chemokines IP-10 and Mig to the antitumoreffects of IL-12. J Leukoc Biol 64:384.

[0127] 33. Koga, S., M. B. Auerbach, T. M. Engeman, A. C. Novick, H.Toma, and R. L. Fairchild. 1999. T cell infiltration into class IIMHC-disparate allografts and acute rejection is dependent on theIFN-gamma-induced chemokine Mig. J Immunol 163:4878.

What is claimed is:
 1. An immunoassay method for detecting MIG, a Th−1cytokine that induces MIG expression, or cells which secrete saidcytokine that induces MIG expression, comprising: a. obtaining a tissuesample from a mammalian host which has been exposed to an immunoreactivesubstance; b. culturing said tissue sample in the presence of saidimmunoreactive substance; c. detecting MIG expression in said sample;and d. correlating said detection to the presence of said cytokine whichinduces MIG expression or cells that secrete said cytokine that inducesMIG expression.
 2. The method of claim 1, wherein said mammalian host isa human.
 3. The method of claim 1, wherein said mammalian host is anon-human primate.
 4. The method of claim 1, wherein said mammalian hostis murine.
 5. The method of claim 1, wherein said mammalian host isporcine.
 6. The method of claim 1, wherein said mammalian host isbovine.
 7. The method of claim 1, wherein said immunoreactive substanceis an antigen or antigenic peptide.
 8. The method of claim 1, whereinsaid tissue sample includes mononuclear cells.
 9. The method of claim 1,wherein said tissue sample is whole blood.
 10. The method of claim 1,wherein said cytokine is IFN-γ
 11. The method of claim 1, wherein saidexposure is natural.
 12. The method of claim 1, wherein said exposure isexperimental.
 13. The method of claim 1 wherein said MIG expression isamplified due to the production of IFN-γ.
 14. The method of claim 1,wherein said MIG expression is IFN-γ mediated.
 15. The method of claim1, wherein said MIG expression is antigen-specific.
 16. The method ofclaim 1, wherein said MIG expression is genetically-restricted.
 17. Themethod of claim 1, wherein said MIG expression is mediated by CD8⁺ Tcells.
 18. The method of claim 1, wherein said MIG expression ismediated by CD4⁺ T cells.
 19. The method of claim 1, wherein said MIGexpression is mediated by NK cells.
 20. The method of claim 1, whereinsaid exposure is in vitro.
 21. The method of claim 1, wherein saidexposure is in vivo.
 22. The method of claim 1, wherein said detectionis accomplished by ELISPOT.
 23. The method of claim 1, wherein saiddetection is accomplished by a flow based system.
 24. The method ofclaim 1, wherein said detection is accomplished by flow cytometry. 25.The method of claim 1, wherein said detection is accomplished by abead-based assay.
 26. The method of claim 1, wherein said detection isaccomplished by RT/PCR.
 27. The method of claim 1, wherein saiddetection is accomplished by ELISA.
 28. The method of claim 1, whereinsaid tissue sample is cultured for less than 20 hours.
 29. The method ofclaim 28, wherein said tissue sample cultured for 4-16 hours.
 30. Themethod of claim 29, wherein said tissue sample is cultured for 16-20hours.
 31. The method of claim 29, wherein said sample includesperipheral blood mononuclear cells isolated from blood drawn from ahuman subject.
 32. The method of claim 1, wherein said sample includesperipheral blood mononuclear cells isolated from blood drawn from ahuman subject.
 33. An immunoassay method for detecting MIG, a Th−1cytokine that induces MIG expression, or cells which secrete saidcytokine that induces MIG expression, comprising: a. obtaining abiological sample from a mammalian host which has been exposed to animmunoreactive substance; b. detecting MIG expression in said sample;and c. correlating said detection to the presence in said sample of saidcytokine which induces MIG expression or cells that secrete saidcytokine that induces MIG expression.
 34. The method of claim 33,wherein said biological sample is plasma, serum, tears, nasalsecretions, or saliva.
 35. The method of claim 33, wherein saidmammalian host is a human.
 36. The method of claim 33, wherein saidmammalian host is a non-human primate.
 37. The method of claim 33,wherein said mammalian host is murine.
 38. The method of claim 33,wherein said mammalian host is porcine.
 39. The method of claim 33,wherein said mammalian host is bovine.
 40. The method of claim 33,wherein said immunoreactive substance is an antigen or antigenicpeptide.
 41. The method of claim 33, wherein said tissue sample includesmononuclear cells.
 42. The method of claim 33, wherein said tissuesample is whole blood.
 43. The method of claim 33, wherein said cytokineis IFN-γ
 44. The method of claim 33, wherein said exposure is natural.45. The method of claim 33, wherein said exposure is experimental. 46.The method of claim 33, wherein said MIG expression is amplified due tothe production of IFN-γ.
 47. The method of claim 33, wherein said MIGexpression is IFN-γ mediated.
 48. The method of claim 33, wherein saidMIG expression is antigen-specific.
 49. The method of claim 33, whereinsaid MIG expression is genetically-restricted.
 50. The method of claim33, wherein said MIG expression is mediated by CD8⁺ T cells.
 51. Themethod of claim 33, wherein said MIG expression is mediated by CD4⁺ Tcells.
 52. The method of claim 33, wherein said MIG expression ismediated by NK cells.
 53. The method of claim 33, wherein said exposureis in vitro.
 54. The method of claim 33, wherein said exposure is invivo.
 55. The method of claim 33, wherein said detection is accomplishedby ELISPOT.
 56. The method of claim 33, wherein said detection isaccomplished by a flow based system.
 57. The method of claim 33, whereinsaid detection is accomplished by flow cytometry.
 58. The method ofclaim 33, wherein said detection is accomplished by a bead-based assay.59. The method of claim 33, wherein said detection is accomplished byRT/PCR.
 60. The method of claim 33, wherein said detection isaccomplished by ELISA.
 61. A method of assessing the effectiveness of acompound or system in inducing an immune response comprising: a.obtaining a tissue sample from a mammalian host which has been exposedto an immunoreactive substance; b. culturing said sample in the presenceof said immunoreactive substance; and c. detecting the induction of theimmune response of said host by up-regulation of MIG expression.
 62. Themethod of claim 61, wherein said system is a vaccine.
 63. The method ofclaim 61, wherein said system is a device used for administering animmunoreactive substance to said mammalian hosts.
 64. The method ofclaim 61, wherein said tissue sample includes mononuclear cells.
 65. Themethod of claim 61 wherein said MIG expression is amplified due to theproduction of IFN-γ.
 66. The method of claim 61, wherein said inductionis mediated by a Th−1 cytokine
 67. The method of claim 61, wherein saidinduction is IFN-γ mediated.
 68. The method of claim 61, wherein saidinduction is antigen-specific.
 69. The method of claim 61, wherein saidinduction is genetically-restricted.
 70. The method of claim 61, whereinsaid MIG expression is mediated by CD8⁺ T cells.
 71. The method of claim61, wherein said MIG expression is mediated by CD4⁺ T cells.
 72. Themethod of claim 61, wherein said MIG expression is mediated by NK cells.73. The method of claim 61, wherein said exposure is in vitro.
 74. Themethod of claim 61, wherein said exposure is in vivo.
 75. A method ofassessing the effectiveness of a compound or system in inducing animmune response comprising: obtaining a tissue sample from a mammalianhost which has been exposed to an immunoreactive substance; culturingsaid sample in the presence of said immunoreactive substance. detectingthe induction of the immune response of said host by detecting thepresence of a chemokine whose production is production is upregulated byIFN-γ.
 76. The method of claim 75, wherein said induction isantigen-specific.
 77. The method of claim 75, wherein said induction isgenetically-restricted.
 78. The method of claim 75, wherein saidchemokine expression is mediated by CD8⁺ T cells.
 79. The method ofclaim 75, wherein said chemokine expression is mediated by CD4⁺ T cells.80. The method of claim 75, wherein said chemokine expression ismediated by NK cells.
 81. The method of claim 75, wherein said exposureis in vitro.
 82. The method of claim 75, wherein said exposure is invivo.
 83. An immunoassay for detecting a Th−1 cytokine which upregulatesthe production of MIG and which is produced in a specific response to animmunoreactive substance, comprising: a. exposing a mammalian host tosaid immunoreactive substance; b. obtaining a sample of plasma or serumfrom host; c. detecting the presence of MIG in said plasma or serum andcorrelating the presence of MIG in said plasma or serum to said host'sproduction said cytokine in specific response to said immunoreactivesubstance.
 84. The immunoassay of claim 83, wherein said cytokine isIFN-γ.
 85. The immunoassay of claim 83, wherein said immunoreactivesubstance is an antigen or antigenic peptide.
 86. The immunoassay ofclaim 83, wherein said host is a human.
 87. The immunoassay of claim 83,wherein said host is a non-human primate.
 88. The immunoassay of claim83, wherein said host is bovine or porcine.
 89. The immunoassay of claim83, wherein said host is murine.
 90. The method of claim 83, whereinsaid detection is accomplished by a flow based system.
 91. The method ofclaim 83, wherein said detection is accomplished by flow cytometry. 92.The method of claim 83, wherein said detection is accomplished byRT/PCR.
 93. The method of claim 83, wherein said detection isaccomplished by ELISA.
 94. An immunoassay method for detecting IFN-γ orIFN-γ-secreting cells, comprising: a. obtaining a tissue sample from ahost which has been exposed to an immunoreactive substance; b. culturingsaid sample in the presence of said immunoreactive substance; c.detecting expression in said sample of a chemokine whose production isupregulated by IFN-γ; and c. correlating said detection to the presenceof IFN-γ or IFN-γ-secreting cells in said sample.
 95. The immunoassay ofclaim 94, wherein said immunoreactive substance is an antigen orantigenic peptide.
 96. The immunoassay of claim 94, wherein said host isa human.
 97. The immunoassay of claim 94, wherein said host is anon-human primate.
 98. The immunoassay of claim 94, wherein said host isbovine or porcine.
 99. The immunoassay of claim 94, wherein said host ismurine.
 100. The method of claim 94, wherein said detection isaccomplished by a flow based system.
 101. The method of claim 94,wherein said detection is accomplished by flow cytometry.
 102. Themethod of claim 94, wherein said detection is accomplished by RT/PCR.103. The method of claim 94, wherein said detection is accomplished byELISA.
 104. A method of monitoring the immunoresponsiveness of a mammal,comprising: obtaining a biological sample from a subject that has beenexposed to a known immunoreactive substance; detecting MIG expression insaid sample; and correlating said detection to the presence in saidsample of cells which secrete a cytokine that upregulates the productionof MIG and thus to the immunoresponsiveness of said mammal.
 105. Themethod of claim 104, further comprising the step of culturing saidsample in the presence of said known immunoreactive substance beforesaid detecting step.
 106. The method of claim 104, wherein said cytokineis IFN-γ.
 107. The method of claim 104, wherein said mammal is a human.108. The method of claim 104, wherein said mammal is a non-humanprimate.
 109. The method of claim 104, wherein said mammal is murine.110. The method of claim 104, wherein said mammal is bovine or porcine.111. The method of claim 104, wherein said immunoreactive substance isan antigen or antigenic peptide.
 112. A method of monitoring theimmunoresponsiveness of a mammal, comprising: obtaining a tissue samplefrom a subject that has been exposed to a known immunoreactivesubstance; detecting in said sample expression of a chemokine whoseproduction is upregulated by IFN-γ; and correlating said detection tothe presence of cells which secrete IFN-γ and thus to theimmunoresponsiveness of said mammal.
 113. The method of claim 112,further comprising the step of culturing said sample in the presence ofsaid known immunoreactive substance before said detecting step.
 114. Themethod of claim 112, wherein said mammal is a human.
 115. The method ofclaim 112, wherein said mammal is a non-human primate.
 116. The methodof claim 112, wherein said mammal is murine.
 117. The method of claim112, wherein said mammal is bovine or porcine.
 118. The method of claim112, wherein said immunoreactive substance is an antigen.
 119. A methodof monitoring the immunoresponsiveness of a mammal, comprising:obtaining a biological fluid sample from a subject that has been exposedto a known immunoreactive substance; detecting MIG in said biologicalfluid sample; and correlating said detection to the immunoresponsivenessof said mammal.
 120. The method of claim 119, wherein saidimmunoreactive substance is an antigen or antigenic peptide.
 121. Themethod of claim 119, wherein said mammal is a human.
 122. The method ofclaim 119, wherein said mammal is a non-human primate.
 123. The methodof claim 119, wherein said mammal is a murine.
 124. The method of claim119, wherein said mammal is porcine or bovine.
 125. An immunoassay kitfor detecting in a sample a Th−1 cytokine which upregulates theproduction of MIG, or cells that secrete said cytokine, comprising: a. abiological sample; b. an immunoreactive substance; c. an indicator forMIG; and d. a detector which detects said indicator.
 126. The kit ofclaim 125, wherein said detector detects MIG by ELISPOT.
 127. The kit ofclaim 125, wherein said detector detects MIG by a flow based system.128. The kit of claim 125, wherein said detector detects MIG by flowcytometry.
 129. The kit of claim 125, wherein said detector detects MIGby RT/PCR.
 130. The kit of claim 125, wherein said detector detects MIGby ELISA.
 131. The kit of claim 125, wherein said detector detects MIGby RIA.
 132. The kit of claim 125, wherein said immunoreactive substanceis DNA.
 133. The kit of claim 125, wherein said immunoreactive substanceis RNA.
 134. The kit of claim 125, wherein said immunoreactive substanceis a protein.
 135. The kit of claim 125, wherein said immunoreactivesubstance is a peptide.
 136. The kit of claim 125, wherein said cytokineis IFN-γ.
 137. An immunoassay kit for detecting IFN-γ or IFN-γ secretingcells in a sample comprising: a. a biological sample; b. animmunoreactive substance; c. an indicator for a chemokine whoseproduction is upregulated by IFN-γ; and d. a detector which detects saidindicator.
 138. The kit of claim 137, wherein said detector detectsIFN-γ by ELISPOT.
 139. The kit of claim 137, wherein said detectordetects IFN-γ by a flow based system.
 140. The kit of claim 137, whereinsaid detector detects IFN-γ by flow cytometry.
 141. The kit of claim137, wherein said detector detects IFN-γ by RT/PCR.
 142. The kit ofclaim 137, wherein said detector detects IFN-γ by ELISA.
 143. The kit ofclaim 137, wherein said detector detects IFN-γ by RIA.
 144. The kit ofclaim 137, wherein said immunoreactive substance is DNA.
 145. The kit ofclaim 137, wherein said immunoreactive substance is RNA.
 146. The kit ofclaim 137, wherein said immunoreactive substance is a protein.
 147. Thekit of claim 137, wherein said immunoreactive substance is a peptide.148. An immunoassay for detecting IFN-γ, or cells which secrete IFN-γ,comprising: obtaining a sample including peripheral blood mononuclearcells from a human host which has been exposed to an immunoreactivepeptide; culturing said sample for a period of 4 to 16 hours in thepresence of said peptide; detecting MIG expression in said sample viaflow cytometry; and correlating said detection to the presence in saidsample of IFN-γ or cells that secrete IFN-γ ; wherein said expression ofMIG is genetically and specifically restricted, was induced by saidIFN-γ, and said induction was mediated by CD8⁺ cells.
 149. A method ofassessing the effectiveness of a compound or system in inducing animmune response comprising: obtaining a sample including peripheralblood mononuclear cells from a human host which has been exposed to animmunoreactive peptide; culturing said sample for 4 to 16 hours in thepresence of said peptide. detecting, via flow cytometry, the inductionof the immune response of said human host by up-regulation of MIGexpression; wherein said expression of MIG is genetically andspecifically restricted, was induced by IFN-γ, and said induction wasmediated by CD8⁺ cells.
 150. An immunoassay for detecting IFN-γ producedin a specifically and genetically restricted response to an antigen orantigenic peptide, comprising: exposing a human host to said antigen orantigenic peptide; obtaining a sample of plasma or serum from host; anddetecting, via flow cytometry, the presence of MIG in said sample andcorrelating the presence of MIG in said sample to said host's productionof IFN-γ in specific response to said antigenic peptide.
 151. A methodof monitoring the immunoresponsiveness of a human, comprising: obtaininga sample including peripheral blood mononuclear cells from a humansubject that has been exposed to a known antigen or antigenic peptide;culturing said sample in the presence of said known antigen or antigenicpeptide for 4 to 16 hours; detecting, via flow cytometry, MIG expressionin said cultured sample; and correlating said detection to the presenceof IFN-γ secreting cells in said sample and thus to theimmunoresponsiveness of said human subject; wherein said expression ofMIG is genetically and specifically restricted, was induced by IFN-γ,and said induction was mediated by CD8⁺ cells.
 152. A method ofmonitoring the immunoresponsiveness of a human subject, comprising:obtaining a sample of serum or plasma from a human subject that has beenexposed to a known antigen or antigenic peptide; detecting MIG in saidsample; and correlating said detection to the immunoresponsiveness ofsaid human subject.
 153. An immunoassay kit for detecting IFN-γ or IFN-γsecreting cells in a sample comprising: a. a sample including peripheralblood mononuclear cells; b. an antigen or antigenic peptide; c. afluorescently labeled antibody to MIG; and d. a detector which detectssaid fluorescently labeled antibody via flow cytometry.
 154. Animmunoassay kit for detecting in a sample a Th−1 cytokine whichupregulates the production of a chemokine, or cells that secrete saidcytokine, comprising: a. an indicator for said chemokine; and b. adetector which detects said indicator.
 155. The kit of claim 154,wherein said Th−1 cytokine is IFN-γ.
 156. The kit of claim 154, whereinsaid chemokine is MIG.
 157. The kit of claim 156, wherein said Th−1cytokine is IFN-γ.
 158. The kit of claim 154, wherein said indicatorincludes an antibody to said chemokine.
 159. An immunoassay fordetecting IFN-γ, or cells which secrete IFN-γ, comprising: obtaining asample including peripheral blood mononuclear cells from a human hostwhich has been exposed to an immunoreactive peptide; culturing saidsample for a period of 4 to 16 hours in the presence of said peptide;detecting MIG expression in said sample via flow cytometry; andcorrelating said detection to the presence in said sample of IFN-γ orcells that secrete IFN-γ; wherein said expression of MIG is geneticallyand specifically restricted, was induced by said IFN-γ, and saidinduction was mediated by CD4⁺ cells.
 160. A method of assessing theeffectiveness of a compound or system in inducing an immune responsecomprising: obtaining a sample including peripheral blood mononuclearcells from a human host which has been exposed to an immunoreactivepeptide; culturing said sample for 4 to 16 hours in the presence of saidpeptide. detecting, via flow cytometry, the induction of the immuneresponse of said human host by up-regulation of MIG expression; whereinsaid expression of MIG is genetically and specifically restricted, wasinduced by IFN-γ, and said induction was mediated by CD4⁺ cells.
 161. Amethod of monitoring the immunoresponsiveness of a human, comprising:obtaining a sample including peripheral blood mononuclear cells from ahuman subject that has been exposed to a known antigen or antigenicpeptide; culturing said sample in the presence of said known antigen orantigenic peptide for 4 to 16 hours; detecting, via flow cytometry, MIGexpression in said cultured sample; and correlating said detection tothe presence of IFN-γ secreting cells in said sample and thus to theimmunoresponsiveness of said human subject; wherein said expression ofMIG is genetically and specifically restricted, was induced by IFN-γ,and said induction was mediated by CD4⁺ cells.
 162. An immunoassaymethod for detecting a chemokine, a Th−1 cytokine that inducesexpression of said chemokine, or cells which secrete said Th−1 cytokinethat induces expression of said chemokine, comprising: obtaining atissue sample from a mammalian host which has been exposed to animmunoreactive substance; culturing said tissue sample in the presenceof said immunoreactive substance; detecting expression of said chemokinein said sample; and correlating said detection to the presence of saidTh−1 cytokine which induces expression of said chemokines, or cells thatsecrete said cytokine that induces expression of said chemokine.